Cell-Free Scaled Production and Adjuvant Addition to a Recombinant Major Outer Membrane Protein from Chlamydia muridarum for Vaccine Development

All animal studies were performed at the University of California, Irvine, in Public Health Service (PHS)-assured facilities in accordance with the guidelines set by the Institutional Animal Care and Use Committee.

1. Glassware preparation

NOTE: All materials used in producing vaccine-grade formulations for animals are endotoxin-free.

1. To destroy contaminating endotoxin, bake cleaned glassware that will hold buffers in an oven at 180 °C for 4 h.

2. Buffer preparation

1. Prepare 250 mL of the Ni affinity purification buffers listed in Table 1. Store them at 4 °C for up to 6 months.

3. Reaction preparation

1. Weigh out 20 mg of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) into an endotoxin-free, 1.5 mL centrifuge tube. Dissolve it in 1 mL of endotoxin-free water, probe-sonicate at least four times at 6 A for 1 min, with 1 min pauses in between, until clear. Remove any contaminant metal from the probe by centrifugation at 13,000 × g for 2 min at 22 °C and then transfer the solubilized lipid to a new 1.5 mL endotoxin-free tube.
2. Weigh out 1 mg of PEG5k-CA8 telodendrimer into a 1.5 mL endotoxin-free tube. Dissolve in endotoxin-free water to a concentration of 20 mg/mL. Vortex until fully dissolved and dilute to 2 mg/mL.
3. In a new endotoxin-free tube, combine 210 µL of 20 mg/mL DMPC solution with 210 µL of 2 mg/mL telodendrimer solution.

4. Cell-free production of MOMP-tNLPs for subunit vaccine formulations

1. Prepare MOMP-tNLPs using cell-free methods modified from a previously published protocol⁵.
   1. Two hours before setting up the cell-free reaction, open the prokaryotic cell-free protein expression kit and thaw one of the reconstitution buffers. Once thawed, add one tablet of EDTA-free protease inhibitor cocktail and let it dissolve fully.
2. Follow this protocol using a kit designed to run 5 x 1 mL reactions.
   ​NOTE: A typical scale-up production is 3 x 1 mL.
   1. For each 1 mL reaction, add 525 µL of reconstitution buffer to the E. coli lysate bottle and gently roll to dissolve. Add 250 µL of reconstitution buffer to the bottle containing reaction additives (e.g., ATP, GTP) and gently roll to dissolve.
   2. Add 8.1 mL of reconstitution buffer to the reaction feed bottle, recap with a rubber stopper (take care not to touch the inside of the rubber stopper), and invert/roll gently to dissolve.
   3. Add 3 mL of reconstitution buffer to the amino acid mixture bottle, recap with a rubber stopper, and invert/roll gently to dissolve.
      NOTE: Take care not to touch the inside of the rubber stopper as this can lead to contamination.
   4. Add 1.8 mL of reconstitution buffer to the methionine bottle, roll gently to dissolve, and then store on ice until use.
3. Prepare the reaction solution.
   1. To the E. coli lysate bottle, add 225 μL of reconstituted Reaction Mix, 270 μL of reconstituted amino acid mix without methionine, and 30 μL of reconstituted methionine. Additionally, add 400 μL of the DMPC/telodendrimer mixture, 15 μg of MOMP plasmid, and 0.6 μg of Δ49ApoA1 plasmid. Roll/gently shake to mix.
      NOTE: Ensure that both plasmids are constructed from the same plasmid backbone. Do not vortex.
   2. Take 20 μL of the total solution and set it aside in a 1.5 mL tube for the GFP-expressing control reaction (see below).
4. Prepare the feed solution. To the feed mix bottle, add 2.65 mL of reconstituted amino acid mix without methionine and 300 μL of reconstituted methionine. Roll/gently shake to dissolve.
   NOTE: At this time, the unused reconstitution buffer and Methionine can be returned to the freezer for storage.
5. Transfer 1 mL of the reaction solution to the inner reaction chamber provided in the cell-free reaction kit and seal when filled. Transfer 10 mL of the feed solution to the outer chamber of the reaction vessel and seal.
   NOTE: Do not overfill the chambers! The presence of air bubbles at the top of both the inner reaction chamber and the inner feed chamber will adversely affect the reaction. Any remaining reaction solution can be placed in a 1.5 mL tube and allowed to mix alongside the main vessel.
6. Add 0.5 μL of the GFP control plasmid (0.5 mg/mL) to the previously aliquoted 20 µL reaction mixture.
   NOTE: Many kits are supplied with a control plasmid for quality-control purposes. Most GFP expressing plasmid with a T7 promoter and E.coli ribosome binding site (RBS) can also be used as the control plasmid.
7. Place the reaction in a shaker at 300 rpm, 30 °C for up to 18 h. To verify that the reaction was successful, use a UV light source to check for fluorescence due to the synthesis of the control GFP (Figure 1A) after as little as 15 min of incubation.
   NOTE: These conditions, particularly temperature, may need to be optimized for expression of other membrane proteins.

5. MOMP-tNLP purification

1. Use immobilized nickel affinity chromatography to purify the MOMP-tNLP nanoparticle complex from the cell-free reaction mixture using the His-tag on the Δ49ApoA1 protein.
   1. Transfer 1 mL of a 50% slurry of His-Tag Purification Resin to a disposable 10 mL chromatography column and equilibrate it with 3 mL of Binding buffer.
   2. Let the buffer drain, cap the outlet, and add 250 μL of Binding buffer to the resin.
   3. Before adding the cell-free reaction to the column, save 20 μL for later analysis by SDS-PAGE. Mix the cell-free reaction with the equilibrated resin and incubate it on a laboratory rocker at 4 °C for 1 h.
2. Uncap the column, wash the cap with 500 μL of additional Binding buffer, and add this liquid to the rest of the column.
3. Collect the liquid flow-through from the column for later analysis by SDS-PAGE.
4. Wash the column with 1 mL of wash buffer containing 20 mM imidazole six times and collect fractions. Take care not to let the resin dry out between washes. On the second wash, vigorously agitate the resin by pipetting up and down using a 1 mL pipette.
5. Elute the MOMP-tNLPs in six 300 μL fractions of Elution buffer 1 (containing 250 mM imidazole), followed by one final elution with 300 μL of Elution buffer 2 (containing 500 mM imidazole). On the second elution, vigorously agitate the resin by pipetting up and down using a 1 mL pipette.

6. Analysis by SDS-PAGE

NOTE: All elution fractions should be analyzed by SDS-PAGE to screen for quantity and purity of the protein of interest.

1. Load 1 µL each of the total lysate and flow-through and then 5 µL for all collected washes and elution fractions.
   1. Mix aliquots of the eluted MOMP–tNLPs, washes, flow-through, and total lysate with 4x SDS-PAGE sample loading buffer. Mix and heat-denature the samples with 10x sample reducing agent unless otherwise indicated.
   2. Analyze the fractions by gel electrophoresis using 1.0 mm, 4 to 12%, Bis-Tris SDS-PAGE gels with 1x MES-SDS running buffer, along with an appropriate molecular weight standard. Run the gels for 35 min at 200 V.
2. Stain the gels according to the manufacturer’s instructions.
   1. Remove the gel from the cassette and place it in 60 mL of gel stain. Microwave the gel in the gel stain for 30 s, and gently rock the container for 30 s to distribute heat evenly. Microwave the gel in the stain to 80–85 °C for another 30 s, and place the gel on an orbital shaker to rock for 5 min.
   2. Microwave the gel a third time for 30 s, and then return to the orbital shaker to rock for another 23 min.
   3. Transfer the gel to a clean container and wash in 100 mL of wash solution (10% methanol, 7% acetic acid) for 30 min.
      NOTE: This is a critical step as it is essential to avoid heating the wash solution. Failure to do so can result in background staining and irregularities in the final gel image.
   4. After washing, rinse the gel in ultrapure water twice for 5 min each.
   5. Image the gels using a gel imager at 600 nm (Figure 2). Use SDS-PAGE to quantify the amount of individual protein in the nanoparticle solution if there is a protein standard for comparison.
      NOTE: In this example, serial dilutions of recombinantly expressed MOMP are resolved by SDS-PAGE and the densities of the bands are quantified using instrument software.
   6. Generate a standard curve using the densities of the MOMP bands. Resolve the MOMP-tNLP samples on the same SDS-PAGE gel and calculate the MOMP component of the particles using the MOMP standard curve (Figure 3).

7. Western and dot blots and storage

1. For western blotting, resolve the samples by SDS-PAGE and transfer the gels using a commercial dry blotting system with standard settings according to the manufacturer’s protocol.
   1. Remove the blots from the stack after the transfer is complete, and incubate each blot overnight at 4 °C in a suitable blocking buffer containing 0.2% Tween 20 and either 0.5 mg/mL MAb40 or 0.2 mg/mL MAbHIS anti-His-tag antibody directed against the His-tag from Δ49ApoA1 protein.
      NOTE: The antibody dilutions used for blotting are 1:1,000 for MAb40 and 1:500–1,000 for MAbHIS antibody.
   2. Wash each blot 3 times for 5 min with PBS-T (1x PBS, 0.2% Tween 20, pH 7.4).
   3. Incubate the blots for 1 h in blocking buffer containing secondary antibody conjugated to a fluorophore (e.g., IRDye) at a 1:10,000 dilution.
   4. Rewash the blots 3 times for 5 min with PBS-T. Use a fluorescence imager to image the blots after the final wash.
2. For dot blots, blot 3 μg of purified MOMP-tNLP and empty tNLP using a dot blot apparatus. Block and develop the blots using the same methods described above for western blotting.

8. Endotoxin assessment

1. Quantify endotoxin levels using an endotoxin testing system based on the Limulus Amebocyte Lysate (LAL) assay. Prepare endotoxin-free 25 mM Tris, pH 7.4, sample buffer using 1 M Tris hydrochloride solution and endotoxin-free water.
   NOTE: Typically, samples need to be diluted using this sample buffer, and the dilutions adjusted to find the suitable range for individual samples. Here, MOMP-tNLP samples are diluted 500-fold in sample buffer and 25 µL are loaded into each well of a device cartridge with 0.05 EU/mL sensitivity. The endotoxin levels of MOMP-tNLP and empty tNLP used in the mouse studies described below are between 0.4 to 12 EU/µg protein depending on the sample.

9. Lyophilization

1. Lyophilize and store the MOMP-tNLP nanoparticles for long-term (up to years) use at -20 °C. To prepare tNLP and MOMP-tNLP suspensions for lyophilization, add trehalose as a protectant during the freezing and lyophilization process.
   NOTE: This process has been extensively validated for a variety of tNLP formulations^7,8.
2. Divide the current volume of the MOMP-tNLP solution by 9 to obtain the volume of 1 M trehalose in sterile, endotoxin-free, deionized water required to reach a final concentration of 0.1 M trehalose. Make note of the final volume and aliquot into endotoxin-free 15 mL or 50 mL polypropylene tubes as desired.
3. Freeze the mixed solution on dry ice and lyophilize it overnight using a lyophilizer. Store the dried formulations at -20 °C until needed.
4. Reconstitute lyophilized tNLPs using endotoxin-free water. Gently roll until the lyophilized cake is fully dissolved and rehydrated. To remove trehalose, dialyze the solution against PBS using a 3.5 kDa cutoff dialysis membrane.

10. Adjuvant addition

NOTE: These and other similar NLP-based sub-unit vaccine formulations can readily incorporate lipophilic adjuvants such as CpG-ODN1826 and FSL-1. CpG-ODN1826 is a modified Class B CpG oligonucleotide (5'-tccatgacgttcctgacgtt-3') with a full phosphorothioate backbone featuring a 5' cholesterol moiety (5'-chol-C6). The conjugation of CpG-ODN1826 to tNLPs is mediated by the hydrophobic interactions between the cholesterol moiety and the phospholipid bilayer of the tNLP and has been demonstrated and well-characterized, as previously reported^9,10.

1. Prior to incorporation into these formulations, purify the cholesterol-modified CpG by reversed-phase chromatography to remove contaminating endotoxin as well as any unmodified CpG molecules.
   1. Upon receipt from the vendor, rehydrate the lyophilized CpG material in endotoxin-free water and purify it on a preparative C4 RP-HPLC column using a separation gradient consisting of 10 mM triethylammonium acetate (TEAA) (mobile phase A) and acetonitrile (mobile phase B).
      NOTE: Additional details are available in Table 2.
   2. Pool and lyophilize the fractions containing cholesterol-modified CpG. To ensure complete removal of residual TEAA, reconstitute the CpG with 15 mL of endotoxin-free water and re-lyophilize it three times.
   3. After the final lyophilization, reconstitute CpG in endotoxin-free water (>20 mg/mL final CpG concentration), aliquot, and store it at -80 °C until needed. For addition to formulations, dilute the CpG to a concentration of 1–2.5 mg/mL.
      NOTE: FSL-1 is available as a vaccine-grade, lyophilized powder. This is reconstituted using sterile and endotoxin-free water at a concentration of 1 mg/mL. The vaccine is administered intramuscularly (i.m.), with each dose containing 10 μg of MOMP in a total volume of 50 μL.
2. To achieve the desired formulation dose, dialyze the nanoparticles into PBS and concentrate them using a centrifugal vacuum concentrator before adjuvant addition. Take care when doing this to prevent complete drying of the sample—check the sample volume every 20–30 min during centrifugation.
3. Add the adjuvant under sterile conditions in a biosafety cabinet. To assess successful incorporation, analyze the final formulations and their components by analytical size-exclusion chromatography (SEC).
   NOTE: For these preparations, an SEC column was used in PBS buffer (0.5 mL/m in flow rate), and elution was detected using a UV-vis diode array detector. Incorporation was assessed by comparing the absorption of the adjuvanted particles to that of the unadjuvanted particles at 214 and 280 nm.
4. Store the adjuvanted MOMP-tNLP and empty tNLP at 4 °C prior to animal use for a period of up to 14 days. To fully assess the stability of a new tNLP formulation, periodically analyze the stored tNLPs by SEC.
   NOTE: Stability will vary from formulation to formulation.

11. Serum testing

1. Obtain female 3-week-old mice (BALB/c, n = 6).
2. Vaccinate the mice intramuscularly (i.m.) in each hindlimb with 10 µg of MOMP in the form of MOMP-tNLP adjuvanted with 5 µg of CpG and 1 µg FSL-1 (total volume per injection = 50 mL).
3. After vaccination, observe the mice until they are able to maintain sternal recumbency.
4. Four weeks after the initial vaccination (prime), vaccinate the animals a second time (boost) with 10 µg of MOMP in the form of MOMP-tNLP adjuvanted with 5 µg of CpG and 1 µg FSL-1 (total volume per injection = 50 mL).
5. On day 56 after the initial vaccination, collect blood to assess antibody titers. Begin by anesthetizing the mice by injecting i.p. a solution of xylazine (0.3 mg/20 g body weight) and ketamine (3.0 mg/20 g body weight). Pinch the front and hind legs to make sure no jerking occurs. Apply petroleum jelly around the eyes to prevent eye dryness during anesthesia.
6. Using a micro-hematocrit capillary tube, puncture the retro-orbital plexus. Collect 100 mL of blood in a microcentrifuge tube.
7. After blood collection, observe the mice until they recover from anesthesia and can maintain sternal recumbency.
8. Let the blood clot at room temperature for 30 min and then spin down at 2,000 × g for 10 min. Collect the serum and freeze at -80 °C.
9. At this time, challenge the animals with Chlamydia muridarum or euthanize them. Euthanize the mice by first injecting i.p. a solution of xylazine (0.3 mg/20 g body weight) and ketamine (3.0 mg/20 g body weight) followed by cervical dislocation.
10. Test serum antibodies specific for MOMP using western blotting techniques as described above. Pool mouse sera from all immunized mice and use the pooled serum in place of a primary antibody at 1:5,000 dilution.